EFFECT OF ELECTR- AND ULTRAPHONOPHORESIS OF THE PHYTOCOMPLEX ON MICROCIRCULATORY AND BIOCHEMICAL PARAMETERS IN PATIENTS WITH KNEE JOINT OSTEOARTHRITIS.

The goal was to study the effect of modulated sinusoidal currents electrophoresis and ultraphonophoresis of the phytocomplex on disruptions in the microcirculation system in the affected joint area and on changes in connective tissue metabolism parameters, metabolic processes, and electrolyte metabolism in patients with knee joint osteoarthritis. Seventy-two patients were randomly assigned to five groups. Patients of the first group were prescribed modulated sinusoidal currents electrophoresis of the phytocomplex. The second group was prescribed ultraphonophoresis of the phytocomplex, the third group was prescribed amplipulse therapy (modulated sinusoidal currents), the fourth group was prescribed ultrasound therapy, and the fifth group was prescribed basic drug therapy. Drug therapy of patients of the fifth group was comparable to the drug treatment of patients of the first four groups. The concentration of the phytocomplex in the working composition was 10%. Electrotherapy was carried out in the full-wave modulated sinusoidal currents mode with I and IV types of operation while ultrasound therapy was carried out in continuous mode with an ultrasound intensity of 0.6 W/cm2. To assess the state of microcirculation, the laser Doppler flowmetry method was used. The pronounced anti-dystrophic effect after the use of modulated sinusoidal currents electrophoresis and ultraphonophoresis of the phytocomplex in patients with knee joint osteoarthritis was based on the correction of microcirculatory disruptions: an increase in the capillary blood flow, an increase in the blood perfusion in tissues, and a decrease in congestion effects in the venular microcirculation. The use of modulated sinusoidal currents electrophoresis of the phytocomplex (ultraphonophoresis of the phytocomplex had an even greater effect) improved the connective tissue metabolism and the content of seromucoid, fibrinogen, and mucoproteins. The use of the studied treatment methods improved magnesium and phosphorus parameters of the electrolyte metabolism. Modulated sinusoidal currents electrophoresis and ultraphonophoresis of the phytocomplex contributed to the elimination of the metabolic imbalance of acid phosphatase. Ultraphonophoresis of the phytocomplex also contributed to balancing of the alanine aminotransferase and alkaline phosphatase content. As a result of the study, the effect of modulated sinusoidal currents electrophoresis and ultraphonophoresis of the phytocomplex on disruptions in the microcirculation system in the affected joint area and on changes in connective tissue metabolism parameters, metabolic processes, and electrolyte metabolism in patients with knee joint osteoarthritis was established. The obtained results provide the basis for further studies to assess the overall effectiveness of the use of modulated sinusoidal currents electrophoresis and ultraphonophoresis of the phytocomplex in patients with knee joint osteoarthritis.

Biotransformation of Progesterone by the Ascomycete Aspergillus niger N402.

The ability of the ascomycete Aspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6ß,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6ß-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.

Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice.

Glaucomas are a major cause of blindness. Visual loss typically involves retinal ganglion cell death and optic nerve atrophy subsequent to a pathologic elevation of intraocular pressure (IOP). Some human glaucomas are associated with anterior segment abnormalities such as pigment dispersion syndrome (PDS) and iris atrophy with associated synechiae. The primary causes of these abnormalities are unknown, and their aetiology is poorly understood. We recently characterized a mouse strain (DBA/2J) that develops glaucoma subsequent to anterior segment changes including pigment dispersion and iris atrophy. Using crosses between mouse strains DBA/2J (D2) and C57BL/6J (B6), we now show there are two chromosomal regions that contribute to the anterior segment changes and glaucoma. Progeny homozygous for the D2 allele of one locus on chromosome 6 (called ipd) develop an iris pigment dispersion phenotype similar to human PDS. ipd resides on a region of mouse chromosome 6 with conserved synteny to a region of human chromosome 7q that is associated with human PDS. Progeny homozygous for the D2 allele of a different locus on chromosome 4 (called isa) develop an iris stromal atrophy phenotype (ISA). The Tyrpl gene is a candidate for isa and likely causes ISA via a mechanism involving pigment production. Progeny homozygous for the D2 alleles of both ipd and isa develop an earlier onset and more severe disease involving pigment dispersion and iris stromal atrophy.

[Synthesis and biological activity of 2,3-secotriterpene acid mono- and diamides].

Four types of amide (C3; C28; C3-C28) conjugates based on 2,3-seco-18alphaH-oleanane and 2,3-secolupane mono- and dicarboxylic acids were synthesized. The range of diamide derivatives was supplemented with C3-C3' and C28-C28' dicondensed amides with two A-secotriterpene backbones educed by reacting monocarboxylic A-secoacids with biogenic amino acid lysine. Compounds with inhibitory action against herpes virus reproduction (EC50 8.7 and 4.1 McM) were found among the synthesized mono- and diamide derivatives containing an ethyl-beta-alaninate fragment. It has been ascertained that diamide with ethyl-beta-alaninate fragment combines anti-herpes virus properties and anti-HIV activity (EC50 5.1 McM). For active compounds, the maximum non-toxic concentration (MNTC)/EC50 ratios ranges from 9.7 to 40.8. The synthesized amide conjugates do not exhibit any marked cytotoxic effects against human tumor cell lines rabdomiosarcoma RD TE32, A549 lung carcinoma and melanoma MS.

[Betulonic amides modified at the ring A by amino acids: synthesis and inhibition of influenza A virus reproduction].

The Beckman rearrangement of carboxy- and alkyloxycarbonylalkylamides of 3-hydroxyiminobetulonic acid led to derivatives of 3a-homo-4-aza-3-oxolup-20(29)-ene and 3,4-seco-2-cyanolupa-4(23),20(29)-diene. An X-ray analysis showed methyl 3-(N-acetoximino)lup-20(29)-enoate is the E-isomer. The compounds synthesized exhibited inhibiting activity toward the reproduction of flu A virus in cell culture.

[Synthesis of dihydroquinopymaric acid conjugates with amino acids].

Synthesis of dihydroquinopymaric acid amides and their 2beta-succinyl and 2beta-phthalyl derivatives containing residues of amino acids was carried out for the first time. Antiviral properties of the compounds synthesized were investigated.

[New betulin derivatives in combination with rimantadine for inhibition of influenza virus reproduction].

The preliminary studies mainly revealed comparable inhibition activities of 3-oxime of betulonic acid, 3beta-O-acetyl-28-O-hemiphthalate of betulin and 3,28-dioxime of betulin against reproduction of influenza viruses A (H1N1), A (H7N1), A (H3N2) and B, as well as against the strains of influenza virus A (H1N1) with intrinsic resistance to rimantadine and A (H7N1) with acquired resistance to the drug. The level of the activity depended on the system used for the virus reproduction. The highest level was observed under conditions providing higher permissibivity, i.e. in the chick embryo fibroblast cell culture for A (H7N1) and in fragments of chick embryo chorioallantoic membranes (for all the viruses). In the experiments with virus A/FPV/Rostock/34 (H7N1) in the chick embryo fibroblast cell culture the average effective concentrations (EC50) of the triterpene compounds were 10.4-17.5 mcM in comparison to EC50 of rimantadine (0.014 mcM). The use of every of the compounds in combination with rimantadine resulted in a 2-16 times decrease of their EC50 and correction of the concentration-effect relation of rimantadine. However, when rimantadine was used alone within the higher range of the nontoxic concentrations (11.6-57.6 mcM). its antiviral properties were significantly less pronounced. As a result the virus titer difference in comparison to the control within the above range of the rimantadine concentrations increased from < 1 to > 2.35 Ig PPU/ml and the relations of the maximal tolerance concentrations of the compounds to their EC50 increased 1.7-15.9 times.

Synthesis and study of antiviral and anti-radical properties of aminophenol derivatives.

A number of sterically-hindered o-aminophenol derivatives have been synthesized, and their antiviral activity in parallel with reactivity towards commonly encountered free-radical intermediates was investigated. Of the compounds tested, the highest activity in suppressing replication of Herpes simplex type l viruses was displayed by N-acyl and N-aryl derivatives of 4,6-di-tert-butyl-2-aminophenol, which were able to interact with organic free radicals and, at the same time, manifested low reactivity towards reactive oxygen species.

Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function.

Glaucoma is a heterogeneous eye disease and a major cause of blindness worldwide. Recently, primary open angle glaucoma (POAG)-associated mutations have been found in the trabecular meshwork inducible glucocorticoid response gene (TIGR), also known as the myocilin gene (MYOC), at the GLC1A locus on chromosome 1q21-q31. These mutations occurred in a subset of patients with juvenile- and adult-onset POAG and exhibited autosomal dominant inheritance. Ocular expression and its involvement in POAG suggest that TIGR/MYOC may have a role(s) in regulating intraocular pressure (IOP). Here, we report the generation and analysis of mice heterozygous and homozygous for a targeted null mutation in Myoc. Our study shows that Myoc mutant mice are both viable and fertile. Our in vivo findings further demonstrate that Myoc is not required for normal IOP or normal ocular morphology. The lack of a discernable phenotype in both Myoc-heterozygous and Myoc-null mice suggests that haploinsufficiency is not a critical mechanism for POAG in individuals with mutations in MYOC. Instead, disease-causing mutations in humans likely act by gain of function.

[Synthesis and antiviral activity of lupane triterpenoids with modified cycle E].

A reductive transformation of the peroxide products of ozonolysis of derivatives of 3beta-O-acetyl-22(17-->28)-abeo-lupa-17(28),20(29)-diene and the subsequent intramolecular ketalization led to a compound with a trioxane fragment. This is a new approach to a skeletal modification of triterpenoid cycle E. An activity of the synthesized compounds was found toward the viruses of type A influenza and herpes simplex.

The mouse anterior chamber angle and trabecular meshwork develop without cell death.

BACKGROUND: The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice. RESULTS: The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation. CONCLUSIONS: These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.

Abnormal levels and minimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells.

The interferon induced, dsRNA-activated, protein kinase, PKR, is a key regulator of translational initiation, playing an important role in the regulation of cell proliferation, apoptosis and transformation. PKR levels correlate inversely with proliferative activity in several human tumor systems. This inverse relationship breaks down in human invasive ductal breast carcinomas which exhibit high levels of PKR (Haines et al., Tumor Biol. 17 (1996) 5-12). Consistent with the data from human tumors, the levels of PKR in several breast carcinoma cell lines, MCF7, T47D, BT20, MDAMB231 and MDAMB468, are paradoxically high compared to those found in the normal breast cell lines MCF10A and Hs578Bst. The activity of affinity- or immuno-purified PKR from MCF7, T47D, and BT20 cells appears to be severely attenuated, as judged by its ability to autophosphorylate, or phosphorylate eIF2 alpha. Furthermore, the activity of the kinase from breast carcinoma cells is refractory to stimulation by dsRNA or heparin. However, PKR from breast carcinoma cells remains functional with respect to its ability to bind dsRNA. The activity of PKR from MCF10A cells is reduced by prior incubation with extracts from MCF7 cells, suggesting that MCF7 extracts contain a transdominant inhibitor of PKR. Deregulation of PKR may therefore provide a mechanism for the development or maintenance of a transformed phenotype of human breast carcinomas, mimicking the effects of manipulation of PKR or eIF2 activity observed in experimental systems. Thus, breast carcinomas may provide the first indication of a role for PKR in the pathogenesis of a naturally occurring human cancer.

Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice.

PURPOSE: To characterize ocular abnormalities associated with iris atrophy in DBA/2J mice and to determine whether mice of this strain develop elevated intraocular pressure (IOP) and glaucoma. METHODS: Different approaches, including slit-lamp biomicroscopy, ophthalmoscopic examination, ultrasound backscatter microscopy, and histology were used to examine the eyes of DBA/2J mice ranging from 2 to 30 months old. IOP was measured in DBA/2J mice of different ages. RESULTS: DBA/2J mice were found to develop pigment dispersion, iris transillumination, iris atrophy, anterior synechias, and elevated IOP. IOP was elevated in most mice by the age of 9 months. These changes were followed by the death of retinal ganglion cells, optic nerve atrophy, and optic nerve cupping. The prevalence and severity of these lesions increased with age. Optic nerve atrophy and optic nerve cupping was present in the majority of mice by the age of 22 months. CONCLUSIONS: DBA/2J mice develop a progressive form of secondary angle-closure glaucoma that appears to be initiated by iris atrophy and the associated formation of synechias. This mouse strain represents a useful model to evaluate mechanisms of pressure-related ganglion cell death and optic nerve atrophy, and to evaluate strategies for neuroprotection.

Intraocular pressure in genetically distinct mice: an update and strain survey.

BACKGROUND: Little is known about genetic factors affecting intraocular pressure (IOP) in mice and other mammals. The purpose of this study was to determine the IOPs of genetically distinct mouse strains, assess the effects of factors such as age, sex and time of day on IOP in specific strain backgrounds, and to assess the effects of specific candidate gene mutations on IOP. RESULTS: Based on over 30 studied mouse strains, average IOP ranges from approximately 10 to 20 mmHg. Gender does not typically affect IOP and aging results in an IOP decrease in some strains. Most tested strains exhibit a diurnal rhythm with IOP being the highest during the dark period of the day. Homozygosity for a null allele of the carbonic anhydrase II gene (Car2n) does not alter IOP while homozygosity for a mutation in the leptin receptor gene (Leprdb) that causes obesity and diabetes results in increased IOP. Albino C57BL/6J mice homozygous for a tyrosinase mutation (Tyrc-2J) have higher IOPs than their pigmented counterparts. CONCLUSIONS: Genetically distinct mouse strains housed in the same environment have a broad range of IOPs. These IOP differences are likely due to interstrain genetic differences that create a powerful resource for studying the regulation of IOP. Age, time of day, obesity and diabetes have effects on mouse IOP similar to those in humans and other species. Mutations in two of the assessed candidate genes (Lepr and Tyr) result in increased IOP. These studies demonstrate that mice are a practical and powerful experimental system to study the genetics of IOP regulation and disease processes that raise IOP to harmful levels.

Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure.

BACKGROUND: Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. RESULTS: Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. CONCLUSIONS: We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

Genetic modification of glaucoma associated phenotypes between AKXD-28/Ty and DBA/2J mice.

BACKGROUND: Glaucoma is a common disease but its molecular etiology is poorly understood. It involves retinal ganglion cell death and optic nerve damage that is often associated with elevated intraocular pressure. Identifying genes that modify glaucoma associated phenotypes is likely to provide insights to mechanisms of glaucoma. We previously reported glaucoma in DBA/2J mice caused by recessive alleles at two loci, isa and ipd, that cause iris stromal atrophy and iris pigment dispersion, respectively. A approach for identifying modifier genes is to study the effects of specific mutations in different mouse strains. When the phenotypic effect of a mutation is modified upon its introduction into a new strain, crosses between the parental strains can be used to identify modifier genes. The purpose of this study was to determine if the effects of the DBA/2J derived isa and ipd loci are modified in strain AKXD-28/Ty. RESULTS: AKXD-28/Ty mice develop glaucoma characterized by intraocular pressure elevation, retinal ganglion loss, and optic nerve excavation. In AKXD-28/Ty, isa causes an iris stromal atrophy phenotype as in DBA/2J. However, the iris pigment dispersion phenotype associated with ipd in DBA/2J does not occur in AKXD-28/Ty. Additionally, a greater severity and speed of retinal and optic nerve damage following intraocular pressure elevation in AKXD-28/Ty compared to DBA/2J mice suggests that AKXD-28/Ty is more susceptible to pressure-induced cell death. CONCLUSIONS: The consequences of the ipd and isa mutations are modified in the AKXD-28/Ty background. These strains provide a resource for the identification of modifier genes that modulate pigment dispersion and susceptibility to pressure-induced cell death.

Antiviral activity of betulin, betulinic and betulonic acids against some enveloped and non-enveloped viruses.

Antiviral properties of betulin, betulinic and betulonic acids were investigated in cell cultures infected with herpes simplex type I, influenza FPV/Rostock and ECHO 6 viruses. All studied triterpenes were active against herpes simplex virus. Betulin and especially betulinic acid also suppressed ECHO 6 virus reproduction.

[Synthesis and antiviral activity of ureides and carbamates of betulinic acid and its derivatives]. / Sintez i protivovirusnaia aktivnost' ureidov i karbamatov betulinovoi kisloty i ee proizvodnykh.

Ureides and carbamates of betulinic acid and its derivatives were prepared in good yields by interaction of betulinic acid, betulonic acid, and betulonic acid 3-oxime with amines, amino acids, and alcohols. Ureides of betulonic acid containing L-Val and L-Met residues were found to be effective against herpes simplex type 1 virus. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Synthesis and antiviral activity of amides and conjugates of betulonic acid with amino acids]. / Sintez i protivovirusnaia aktivnost' amidov i kon''iugatov betulonovoi kisloty s aminokislotami.

Betulonic acid amides with aliphatic and heterocyclic amines and with L-amino acids were synthesized by the acid chloride method. Betulonic acid amide and L-methionine derivatives of betulonic acid and its 3-oxime effectively inhibit the influenza A virus. Betulonic acid octadecylamide is active against the herpes simplex type 1 virus. The conjugate of betulonic acid 3-oxime with L-methionine is also active toward HIV-1. The tested compounds mainly show no activity toward the ECHO6 virus, which is devoid of a coat. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

[Synthesis and antiviral activity of hydrazides and substituted benzalhydrazides of betulinic acid and its derivatives]. / Sintez i protivovirusnaia aktivnost' gidrazidov i zameshchennykh benzal'gidrazidov betulinovoi kisloty i ee proizvodnykh.

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhydrazides, were synthesized. Their antiviral activities toward of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.